The liver performs various functions including the synthesis and secretion of most serum proteins such as albumin and lipoproteins, the synthesis of export lipids coupling to proteins, detoxification, the formation and secretion of bile, blood sugar regulation by the production of sugar, urea synthesis caused by the resulting amino acid degradation, the activation of vitamins, the synthesis and degradation of glycogen, the synthesis of glutathione and metallothionein, and other functions.
Hence, many researchers have cultured liver cells in order to study those functions that are more abundant in liver cells than in other tissue cells, such as their most characteristic ability to synthesize and secrete plasma proteins, and these functions have been utilized for many years.
With the progress of biotechnology, useful substances are actively produced by genetic recombination and cell fusion methods. As a result, animal cell cultivation has become more important than ever. Since the aforementioned abilities of liver cells to synthesize albumin, lipoproteins, export lipids, urea, glycogen, glutathione, metallothionein and the like are more abundant than those of other tissue cells, they are interesting hosts for the production of substance by animal cell cultivation. However, cultivation techniques are not satisfactory at present for maintaining hepatocytes (normal liver cells) capable of producing plasma proteins during cultivation periods.
Normal human hepatocytes are extremely difficult to obtain due to their rapid decrease in viability following autopsy. Additionally, the human liver is one of the few organs in adults capable of regeneration. However, replicative cultures of adult human hepatocytes have never been adequately established, as these cells have a very limited lifespan when put into cell culture.
Many cell lines have serious limitations because they are not of human origin, or are not more closely representative of the normal human liver cell. There are several examples of animal liver cell cultures derived from experimental laboratory animals such as rats (Tsao et. al., Exp. Cell Res., 1984, 154:38-52; Enat et al., Proc. Nat. Acad. Sci USA, 1984, 87: 1411-1415). Rat liver epithelial cells from adult rat liver tissue have been established using serum free medium (Chessebeuf and Padieu, In Vitro, 1984, 20:780-795; Enat et al., Proc. Natl. Acad. Sci., 1984, 81:1411-1415). Rat liver cells have been transformed by transfection with SV40 DNA (Woodworth et al., Cancer Res., 1987, 46: 4018-4026; Ledley et al., Proc. Nat. Acad. Sci. USA, 1987, 84: 5335-5339) but those cells are not suitable for human drug metabolism or carcinogenesis studies because of xenobiotic metabolism differences between rat and human liver cells. Clonally-derived cultures of human hepatocytes have been reported (Kaighn and Prince, Proc. Nat. Acad. Sci., 1971, 68:2396-2400).
Long-Term cultures of human fetal liver have been established (Salas-Prato, M. et al., In Vitro Cell Dev. Biol., 1988, 24:230-238; Sells, M. A. et al., In Vitro Cell Dev. Biol., 1985, 21:216-220); however, the inherent differences between fetal and adult liver, especially in the area of xenobiotic metabolism make adult hepatocytes a more suitable model for carcinogenesis and toxicity studies, for example. Rat liver epithelial cells from adult rat liver tissue have been established using serum-free medium (Chessebeuf and Padieu, In Vitro, 1984, 20:780-795; Enat et al., Proc. Natl. Acad. Sci., 1984, 81:1411-1415).
For the purpose of obtaining substitutes for hepatocytes, studies have been actively conducted to create hepatoma cell-derived cell lines that have characteristics peculiar to hepatocytes (normal liver cells) and which are capable of producing liver-specific proteins such as albumin. Hepatoma cells have high proliferation potency and are therefore promising hosts for the production of liver substances. Human hepatoma cell lines have been cultured and are available (e.g., Knowles et al., U.S. Pat. No. 4,393,133, issued Jul. 12, 1983; Knowles B. B. et al., Science, 1980, 209:497-499; Monjardino J. and Crawford E., Virology, 1979, 96:652-655; Park J. G. et al., Int. J. Cancer, 1995, 62:276-282; Zhong et al., PNAS, 2005, 102(26):9294-9299; Fu and Cheng, Antimicrobial Agents and Chemotherapy, 2000, 44(12):3402-3407). For example, the human hepatoma cell line, HepG2, is disclosed in U.S. Pat. No. 4,393,133. Further experiments utilizing the HepG2 cell line are reported by Kelly et al., In Vitro Cell. and Dev. Biol., 1989, 25:217-222; U.S. Pat. No. 5,290,684; and Darlington et al., In Vitro Cell. and Dev. Biol., 1987, 2-3:349-354. The human hepatoma cell line, HuH-7, is discussed in Nakabayashi et al., Cancer Research, 1982, 42:3858-3863.
Known human-derived hepatoma cell lines include, but are not limited to: HLF (Okayama University, medical school: 1975), HLE, c-1 (Okayama University, medical school: 1975), HuH-6 clone 5 (Okayama University, medical school: 1976), HuH-7 (Okayama University, medical school: 1979), C-HC-4 (Hokkaido University, school of medicine: 1979), HCC-M (Keio University, school of medicine: 1980), JHH-1 (The Tokyo Jikei University School of Medicine: 1980), JHH-2 (The Tokyo Jikei University School of Medicine: 1982), JHH-4 (The Tokyo Jikei University School of Medicine: 1983), KIM-1 (Kurume University, school of medicine: 1983), JHH-5 (The Tokyo Jikei University School of Medicine: 1984), JHH-6 (The Tokyo Jikei University School of Medicine: 1984), OHR (Showa University, school of medicine: 1985), KMCH-1 (Kurume University, school of medicine: 1985), KMG-A (Kurume University, school of medicine: 1985), JHH-7 (The Tokyo Jikei University School of Medicine: 1986), JHC-1 (The Tokyo Jikei University School of Medicine: 1986), KYN-1 (Kurume University, school of medicine: 1986), KYN-2 (Kurume University, school of medicine: 1987), HCC-T (Keio University, school of medicine: 1986), HPT-NT/D3 (Kyushu University, faculty of medicine: 1986), Hep-tabata (Mie University, Faculty of Medicine: 1986), HuCC-T1 (Toyama Medicine and Pharmaceutical University, faculty of medicine: 1987), HuH-28 (Okayama University, medical school: 1987). See HUMAN CELL, Vol. 1, No. 1, p. 106-126, 1988.
Aggressive attempts have also been made to prepare artificial livers by culturing liver cells using bioreactors. The use of artificial support devices has had a dramatic effect on kidney, heart and lung transplantation. Bio-artificial livers (BAL) and other supportive systems for long-term preservation of liver functions have been described (see, for example, Anand A. C., Indian J. Gastroenterol., 2003, 22 Suppl 2:S69-74; Ueda et al., ASAIO J, 2003, 49(4):401-6; Tilles et al., J. Hepatobiliary Pancreat. Surg., 2002, 9(6):686-96; Metab. Brain Dis., 2005, 20(4):327-35; and Park and Lee, J. Biosci Bioeng., 2005, 99(4):311-9. Liver assist devices (LAD) have been described previously (see, for example, Lu et al., Tissue Eng., 2005, 11 (11-12):1667-77; Pless and Sauer, Transplant Proc., 2005, 37(9):3893-5; Millis and Losanoff, Nat. Clin. Pract. Gastroenterol Hepatol., 2005, 2(9):398-405; and George J., J. Assoc. Physicians India, 2004, 52:719-22). Such LAD could find application in a number of transplant situations. For example, an artificial liver or LAD could allow patients in fulminant hepatic failure to be stabilized and calmly evaluated before operating; it could stabilize and assist patients after transplant, particularly in situations in which the graph fails to respond on reperfusion; and in some instances it may serve as a substitute for transplant. The device would allow time for the patient's natural liver to regenerate, sparing the expense of operation, the life-long dependence on immunosuppression and the likelihood of premature mortality.
To facilitate the practical use of BAL and LAD, it is desirable to develop a light and small-sized bioreactor apparatus and its peripheral devices. To this end, the creation of cell lines capable of producing the necessary and sufficient amounts of liver-specific proteins in small amounts of media is desired. Human hepatoma cell lines are potentially useful in such systems due to the shortage of normal donors. Investigators have produced a hepatoma cell line potentially useful in BAL systems by overexpressing bcl-2, an anti-apoptosis gene, in hepatoma HepG2 cells (Terada S., J. Biosci. Bioeng., 2003, 95(2):146-51).
Liver cancer is a very common malignancy. Most liver cancers are caused by viral hepatitis viruses, such as hepatitis B virus (HBV) and hepatitis C virus (HCV). Currently, there is no effective therapy.
HCV infection is a major human infectious disease for which there is no effective vaccine. Despite intensive research, the nature of HCV protective immunity is still not well defined. Many lines of evidence suggest the importance of cellular immunity to clear viral infection, but the role of antibody-mediated responses in HCV infection is not known. Most patients with HCV infection have antibodies against viral antigens, but the proportion and strength of the neutralizing antibodies in patient sera are unknown. One limitation to evaluate the nature of neutralizing antibodies is the lack of an infectious cell culture system or small animal model that supports viral infection. Recent success on the cell culture system for one HCV isolate (genotype 2a) has provided a valuable tool to investigate humoral immunity in HCV. It would be advantageous to have available a well-differentiated human hepatoma cell line in which HCV robustly replicates and causes overt cytopathic effects as in nature.
Although there are cell lines derived from human liver cancer, most or all are poorly differentiated and exhibit few liver cell functions. These cell lines are not representative of human tumors. In addition, there is not an efficient cell culture system to culture HBV and HCV, which has dramatically hampered the discovery of antiviral drugs. Although several groups have successfully used primary hepatocyte culture systems and detected low-level replication of HCV, for example, an efficient culture system to grow wild-type HCV has been difficult to achieve (Tagawa M. et al., J. Gastroenterol Hepatol, 1995, 10:523-527; Ito T. et al., J. Gen. Virol., 1996, 77 (Pt. 5):1043-1054; Ito T. et al., Hepatology, 2001, 34:566-572). In 2005, several groups reported the success of cell culture systems that support full HCV production for one particular strain, JFH1, a genotype 2a isolate (Heller T. et al., Proc. Natl. Acad. Sci. USA, 2005, 102:2579-2583; Wakita T. et al., Nat. Med., 2005, 11(7):791-796; Zhong J. et al., PNAs, 2005, 102(26):9294-9; Lindenbach B. D. et al., Science 2005, 309(5734):623-6).
Currently, the only animal model for HCV infection is the chimpanzee (Lanford R. E. et al., Virology, 2002, 293:1-9). On of the primary advantages of the chimpanzee is that it represents a true infection model, though viral pathogenesis may not be identical to human infection (Alter M. J. et al., N. Eng. J. Med., 1999, 341:556-562; Bassett S. E. et al., J. Virol., 1998, 72:2589-2599). The model has provided tremendous knowledge about the host immune responses to HCV infection. The major problems associated with using the chimpanzee model are availability, cost, and resources. Tree shrews (tupaias) are small animals closely related to primates, which adapt easily to a laboratory environment. It has been reported that the tree shrew, Tupaia belangeri, is susceptible to infection with a variety of human viruses in vivo, including hepatitis viruses (die Z. C. et al., Virology, 1998, 244:513-520).
Transgenic mice that express HCV-core protein have been used to study liver pathology and carcinogenesis (Lemon S. M. et al., Trans. Am. Clin. Climatol. Assoc., 2000, 111:146-156). Most of the transgenic lines did not show immediate toxic effect on liver cells, while one transgenic animal line showed lymphocytic infiltration with hepatocyte necrosis (Zhao X. et al., J. Clin. Invest., 2002, 109:221-232). Two other lines showed steatosis and hepatocellular carcinoma (Moriya K. et al., J. Gen. Virol., 1997, 78 (Pt. 7):1527-1531; Moriya K. et al., Nat. Med., 1998, 4:1065-1067). Studies using HBV transgenic mouse models have provided significant insights into HBV immunopathogenesis (Chisari F. V. et al., Science, 1985, 230:1157-1160; Chisari F. V. et al., Hepatology, 1995, 22:1316-1325). However, transgenic models are not yet widely used in studies on HCV immunology. One inherent problem using transgenic mouse models is host immune tolerance to viral proteins, which limits and complicates data interpretation.
It would be advantageous to have available human liver cancer cell lines and animal models that are analogous to the human tumor. Potential applications of such cell lines include, but are not limited to: screening and evaluating anti-tumor drugs; culturing HBV and HCV in a manner resembling the naturally occurring infection; screening and evaluating antiviral drugs; and studying liver cell metabolism.